Process of producing aminobenzoyl amino acids



Patented Jan. 9, 1 951 PROGES S OF BRODUCING AMINOBENZOYL :AMI NO' ACIDS .JohnIHalley Mowat, 'Orangeburg, andCoy Webster Waller, Pearl River, N. Y., assignors to American'Cyanamid Company,New York; N. Y., a'corporation of'Maine This invention relates to an improved method of preparing aminobenzoyl amino acids. More particularly, it relates to the preparation of raminobenzoyl amino acids from the correspond- ,ing nitrobenzoyl amino acids by reduction.

In our copen ding application, Serial No. "771,570, filed August'30, 1947,, of which this is'a continuation-impart, we discosed'certain glutfamic acid amides of para' nitrobenzoic acid, especially those having a polypeptide linkage, such as 'para nitrobenzoylglutamylglutamyl luftamic acid which could be reduced to form th "corresponding amino derivative.

disclosed in our above named c'opending applicat on, the reaction products of para-aminobenzoylpolyglutamic acids with 2,4,5 -triamino- "'fi-hydroxypyrimidine, and alpha, beta-dibromo- 'propionadehyde are growth stimulating "factors for bacteria andhigher forms of'an mal life and efiect' the formation of haemoglobin. These aminobenzoyl polypeptides are important intermediates and it is desirable to have convenient methods of preparin'gthem in'goodyields.

In the past it is known to reduce nitro aliphatic compounds to the corresponding amino aliphatic 'fcompounds using metals such as 'zinc',"-ti'n or iron an'd-a-mineral acid. Oftenthe'se strongacids are deleterious to sensitive compounds-"such asamino acids. When using dilute or weak acids the re- "duction is not complete and yields of' the-desi'red compound are low. We have now found that nitrobenzoyl amino 1 acids can be reducedto the correspondingamino- "benzoyl anino acid by use'of a 'zinc-coppercouple "which produces high yieldsof pure products in a r'elatively short'time. The-general reactionwhich "takes place may be iHustrate'd by thefollowing equationz zinc-copper couple No Drawing. Application; December 31, 1948,v

Serial No. 63,741 1 .2 The zinc-copper couple used'in the reaction of the present invention can be prepared by mixing an aqueous slurry of finely divided zinc with a water soluble salt of copper'such as cupric' acetate or 'cupric sulfate. The copper is plated on the zinc .partic'es and produces a reducing agent which is used as the prepared slurry or as the dried powder in the reduction of nitrobenzoyl- .amino acids to the corresponding aminobenz'oylamino acids.

tures of from about 15 .C. 'to about 75 0.. although we prefer to carry out the reaction at from about 20 C.to ;50 C.

The following examples illustrate in detail. the

application of the invention to thepreparation of representative aminobenzoylamin'o acids.

Example 1 To '31 g. of cupric acetate dissolved in about 2001111501 water is added rapidly a suspension-of '310 g. zinc dust suspendedinabout 500 ml. of water, with vigorous stirring.

The zinc-copper couple is filtered off, Washed with water, alcohol and ether and dried.

copper couple, prepared above, over periodof'2'3.

minutes. The .pH is held constant. The temperature rises to 49 C. The suspension .is

stirred for'fi'l minutes more and the excess zinccopper' filtered off.v A Bratton Marshall amine *a ssay'gives 10013 conversion of nitro compound to amine: compound.

, Example 2 v Gamma-ethyl -p-nitrob-enzoylagammwglutam- -'yl'-gam'rna-glutamylglutamate as a. partial dried *cakerepresent ng 6.25 kg; of dry materialf'is :suspended in 43' liters of water.

This suspension is adjusted to pH. 3.5 and maintained at 'this pH by adding hydrochloric acid while the nitro: group is reduced. 'Chopp'ed ice isadded as neededito keep the temperature at. 30 C. The'nitro group is 2,687,886 T I E 3 dust in 23.5 liters of water fairly rapidly. (The zinc-copper dust suspension is prepared by adding all at once a solution of 350 g. of copper acetate in 3.5 liters of water to a suspension of 3.5 kg. of zinc dust in liters of 'water.) Ten liters of water Was used in completing the transfer of the zinc-copper dust. A Bratton Marshall amine de- 7 termination indicated 93.5% reduction of the nitro to an amine group.

Example 3 To 99 g. of para-nitrobenzoyl-alpha, alpha-- diglutamylglutamic acid tetra-ethyl ester in a solution of 400 ml. of glacial acetic acid and 600 ml. of water, at a temperature of about 33 C., is added a portion of zinc-copper dust. The zinccopper dust seemed to conglomerate and therefore the temperature was raised to 40 C To the mixture is added 250 ml. of ethanol and the temperature raised to 45 C. A further portion of zinc-copper dust making a total of 58 g. is added to the mixture. The mixture is stirred ten minutes longer and filtered. The filtrate is diluted to 2500 ml. with water, cooled and filtered. The precipitate is dissolved in 750 ml. of ethanol and 750 ml. of water added. The mixture was cooled'and filtered. The precipitate is recrystallized several times from ethanol and finall added to a solution of 1000 ml. of water and 100 ml. of acetic acid heated to 90 C. This solution is filtered hot. Upon cooling an almost white precipitate of para-aminobenzoyl-alpha, alpha-diglutamylglutamic acid tetraethyl ester is obtained. The product melted at 148 C. On analysis theproduct checked closely with the theoretical values.

Example 4 A zinc-copper couple is prepared by slurring 16.3 parts finely powdered zinc in 50 volumes of water, slowly adding 1.92 parts CuSOafiI-IzO dissolved in 10 volumes of water, and stirring vigorously for 10 minutes until all of the copper is plated out on the zinc.

Diefhyl p-nitrobenzoylaminomalonate parts) is added to 250 volumes of l N sodium hydroxide and stirred vigorously for 2% hours at 25 to 30 C.; at the end of this time a complete solution is obtained, indicating complete hydrolysis of the ester to p-nitrobenzoylaminomalonic acid. To this solution is then added 30 volumes of 5 N hydrochloric acid, which gives a faint spot on Congo red test paper. While maintaining the temperature at 30 to 35 C. by means ofexternal cooling, the zinc-copper couple as a slurry is added slowly to the solution. of p-nifrobenzoylaminomalonic acid over a minute period; sufficient concentrated (36%) hydrochloric acid is added to maintain a black spot on Congo red test paper. After completion of the addition of the zinc-copper couple, stirring at 30 to 35 C. is continued minutes longer. More concentrated hydrochloric acid is added to maintain the black spot on Congo red test paper; total usage of concentrated hydrochloric acid is 44 volumes. Then 90 volumes of concentrated (28%) ammonia is added to give a strong spot on phenolphthalein test paper; this quantity of ammonia dissolved everything but the unreacted zinc. The solution is filtered and the zinc washed with water; 2 parts of zinc is recovered. The deep yellow reduction filtrate is 590 volumes. Titration of a sample of this filtrate with N/10 sodium nitrite solution showed that the reduction is 98% complete, calculated as vp-aminobenzoylmalonic. acid'present.

Example 5 While maintaining the temperature at 30 to C. a zinccopper slurry (prepared as in the preceding example) is added slowly over a 15 minute period to a slurry of 12.? parts p-nitrobenzoyl-dl-serin'e in 250 volumes of water; sufficient concentrated (36%) hydrochloric acid is added to maintain a black spot on Congo red test paper. Stirring at 30-35 C. is continued for 20 minutes after all of the zinc-copper slurry is added; more concentrated (36%) hydrochloric acid is added to maintain the black spot on Congo red test paper. Then the unreacted zinc is filtered off, the cake is washed well with water, and discarded. The light yellow filtrate was 500 volumes. Titration of a small aliquot of the filtrate with N/10 sodium nitrite solution shows that the reduction is 98% complete, calculated as p-aminobenzoyl-dl-serine present.

Example 6 20 parts of gamma-ethyl-para-nitrobenzoylgamma-glutamylglutamate was suspended in 190 cc. of water and 20 cc. of ethanol and reduced with a zinc-copper couple containing 1'7 parts of zinc and 5 2 part of copper. Hydrochloric acid is used to maintain the pH of 3.5. After allowing the reaction to proceed for about one hour, the excess zinc is filtered oif, ammonium hydroxide added until the filtrate is distinctly alkaline and hydrogen sulfide passed in for 30 to 40 minutes. The zinc sulfide is filtered off, and the filtrate brought to pH 3 and evaporated to dryness using a high vacuum pump and a dry ice trap. Ethanol is added and evaporated again. The solid is taken up in about cc. of absolute ethanol, filtered to remove the ammonium chloride filtrate, evaporated to dryness to give 17.4 g. of a flaky solid. A Bratton-Marshall amine test showed this to be 88% pure. This represents an 84% yield of gamma-ethyl-para-aminobenzoylgamma-glutamylglutamic acid.

Example 7 A zinc-copper couple is made by adding a solution of 0.33 g. of copper acetate in 3.3 ml. of water to a suspension of 3.27 g. of zinc dust in 9.31 ml. of water. The gamma-ethyl, p-nitrobenzoylglutamate (3.23 g.) is suspended in 6'? ml. of water and sodium hydroxide is added to effect solution. Then concentrated hydrochloric acid i used to bring the pH to 3.5. A fine suspension appears throughout the solution. The zinc-copper suspension and hydrochloric acid are added alternately at such a rate that the pH remained at 3.5 and the temperature of the mixture did not go above 35 C. After the addition of the zinccopper, the whole is stirred for an additional 45 minutes or until no more hydrochloric acid is necessar for maintaining the pH. The excess zinc-copper is'filtered 0d and the clear solution is subjected to a Bratton-Marshall amine assay. A yield of of gamma-ethyl, p-aminobenzoylglutamate is obtained.

Example 8 50 g.- of para-nitrobenzoylglutamic acid of 93.1% purity is slurried in I50 cc. of water, heated to 68 C. and a solution containing 0.5 g. of cuprous chloride in 25 g. of water is added. 45 g. ofzinc dust is added while the solution is well stirred. Then concentrated hydrochloric acid is carefullyaddecl to an acid reaction when tested on Congo red paper. When a clear solution is Example 9 Para-nitrobenzoyl-betaPalanine is reduced with zinc-copper couple by the procedure of Example 2 in aqueous solution using a pH of 3.0. The paraaminobenzoyl-beta-alanine obtained has a melting point of 148 C. to 149 C.

Emmple 10 The tetraethyl ester of para-nitrobenzoyl-alpha-glutamyl-gamma-glutamy1glutamic acid is reduced with zinc-copper couple at a pH of 4 in a solution of 50% ethanol. The product obtained has a melting point of 117 0.

Example 11 Following the procedure of Example 2, the following compounds were reduced with zinc-copper couple at a pH of 3-4 in water or aqueous ethanol; para nitrobenzoyl gamma glutamyl alpha glutamylglutamic acid tetraethyl ester; gammaethyl-para-nitrobenzoyl glutamate; para-nitrobenzoyl glutamic acid diamide; para-nitrobenzoyl glutamic acid gamma amide; para-nitrobenzoyl-alpha-gamma-glutamyldiglutamic acid; para nitrobenzoyl alpha -,gamma glutamyl diglutamate tetraethyl ester; para-nitrobenzoylalpha glutamyl gamma glutamylglutamic acid tetraethyl ester; para-nitrobenzoyl-gammaglutamyl leucine; ethyl-para-nitrobenzoyl-gamma-glutamyl glycinate; para-nitrobenzoyl-gamma-glutamyl-beta-alanine; para-nitrobenzoyl aspartic acid; para-nitrobenzoyl-alpha-glutamylglutamic acid; para-nitrobenzoyl-alpha-alanine and para-nitrobenzoyl-dl-threonine. The yields of the corresponding para-aminobenzoyl derivatives were from 80% to 100% of the theoretical value.

It will be understood that various modifications may be made in the specific procedures described without departing from the scope of the invention.

We claim:

1. A method of preparing a member of the group consisting of aminobenzoylamino acids, amides and esters thereof which comprises reacting the corresponding nitrobenzoyl compounds with zinc-copper couple at a pH of about 1 to about 4.5 in the presence of a solvent.

2. A method of preparing aminobenzoylpolyglutamic acid esters which comprises reacting the corresponding nitrobenzoyl compounds with zinccopper couple at a pH of about 1 to about 4.5 in the presence of a solvent.

3. A method of preparing para-aminobenzoylpolyglutamic acid esters which comprises reacting the corresponding para-nitrobenzoy1 compounds with zinc-copper couple at a pH of about 1 to about 4.5 in the presence of a solvent.

4. A method of preparing aminobenzoyltriglutamic acid esters which comprises reacting the corresponding nitrobenzoyl compounds with zinccopper couple at a pH of about 1 to about 4.5 in the presence of a solvent.

5. A method of preparing gamma-ethyl-paraaminobenzoyl gamma-glutarnyl-gamma-glutamylglutamate which comprises reacting the corresponding gamma-ethyl-para-nitrobenzoyl compound with zinc-copper couple at a pH of about 2.5 to about 4.0 in the presence of a substantially aqueous solvent.

6. A method of preparing gamma-ethyl-paraaminobenzoyl-gamma-glutamylglutamate which comprises reacting the corresponding gammaethy1-para-nitrobenzoyl compound with zinccopper couple at a pH of about 2.5 to about 4.0 in the presence of a substantially aqueous solvent.

7. A method of preparing gamma-ethyl-paraaminobenzoyl-glutamate which comprises reacting the corresponding gamma-ethyl-nitrobenzoyl compound with zinc-copper couple at a pH of about 2.5 to about 4.0 in the presence of a substantially aqueous solvent.

8. A method of preparing para-aminobenzoylglutamic acid which comprises reacting the corresponding para-nitrobenzoylglutamic acid with zinc-copper couple at a pH of about 2.5 to about 4.0 in the presence of a substantially aqueous solvent.

JOHN HALLEY MOWAT. COY WEBSTER WALTER.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 19,202 Ellis 1900 1,492,665 Beall May 6, 1924 OTHER REFERENCES Meyer, Beilstein, Handbuch der Org. Chem, 4th ed., vol. 14, p. 358 (1931).

Houben, Die Methoden der Drg. Chem. (3d ed.), vol. 2,10. 304 (1943).

Wagner-Jauregg et al., Chem. Abstracts, vol. 41,1 1). 2118-2119 (1947).

Mowat et al., J. Am. Chem. Soc, vol. '70, pp. 1096-1102 (1948). 

1. A METHOD OF PREPARING A MEMBER OF THE GROUP CONSISTING OF AMINOBENZOYLAMINO ACIDS AMIDES AND ESTERS THEREOF WHICH COMPRISES REACTING THE CORRESPONDING NITROBENZOYL COMPOUNDS WITH ZINC-COPPER COUPLE AT A PH OF ABOUT 1 TO ABOUT 4.5 IN THE PRESENCE OF A SOLVENT. 